Soft ferroelectret ultrasound receiver for targeted peripheral neuromodulation

Bioelectronic medicine is a rapidly growing field where targeted electrical signals can act as an adjunct or alternative to drugs to treat neurological disorders and diseases via stimulating the peripheral nervous system on demand. However, current existing strategies are limited by external battery requirements, and the injury and inflammation caused by the mechanical mismatch between rigid electrodes and soft nerves. Here we report a wireless, leadless, and battery-free ferroelectret implant, termed NeuroRing, that wraps around the target peripheral nerve and demonstrates high mechanical conformability to dynamic motion nerve tissue. As-fabricated NeuroRing can act as an ultrasound receiver that converts ultrasound vibrations into electrostimulation pulses, thus stimulating the targeted peripheral nerve on demand. This capability is demonstrated by the precise modulation of the sacral splanchnic nerve to treat colitis, providing a framework for future bioelectronic medicines that offer an alternative to non-specific pharmacological approaches.

this article, especially if the target tissue is under human skin.Although it has been found that the response from ferroelectrets increases with frequencies, and get good response in the ultrasonic regions (in published articles that the current manuscript ignores), it is very difficult to understand how such a small device under human skin is capable of responding to ultrasonic frequencies with such large voltage response.Along this same line, where are the current plots?Did the authors try to characterize output short circuit current from these devices?Did the authors consider the matching impedance between the device and the instrument used to measure the generated voltage?Obtaining real electro-mechanical measurements from ferroelectrets is not a trivial task, and this reviewer believes that these experiments/studies will generate questions about the electrical voltage output that they are measuring -which could be due to sources other than the ferroelectret.
Finally, although their application may appear fancy, it takes a quick look to the references and relevant prior art to realize that this is also not a novel concept -this reviewer has personally read this same application roughly 5 years ago.Lastly, it's disheartening to note that they not only failed to cite any recent relevant work (including a very recent comprehensive review article on ferro electrets and their applications), but also neglected to reference important studies by other well-known researchers in the field of ferroelectrets.
2. Line 119-121, the authors mentioned that "Remarkably, even at 10 cm under layered tissues including skin, fat and muscle, the non-woven fabric yielded a voltage output of > 250 mV (right, Fig. 2d), still capable of effectively stimulating neural tissue (> 72 mV)."Please give the reference to prove it.
Response: Many thanks for the reviewer's reminder.For greater rigor, references 35 and 36 have been added to support our statement in the revised manuscript.
New added references in revised manuscript: 36.
3. For Supplementary Video 2, it cannot prove that muscle activation is caused by ultrasoundinduced electrical stimulation.Hand pressure also can make the ankle joint move.This animal experiment may be done keeping the leg hanging in the air.

Response:
We are grateful for the reviewer's rigor and professionalism.To avoid ambiguity, we used a frame to fix the ultrasound probe and suspend it in the air, without manual operation (Revised Supplementary Fig. 23a).Moreover, there is an ultrasound coupling agent between the ultrasound probe and the rat skin, which also excludes the impact of the probe's own weight (Revised Supplementary Fig. 23b).Subsequently, as requested by the reviewer, we kept the rat's leg hanging in the air before applying ultrasound (Revised Supplementary Fig. 23a).Finally, after applying ultrasound, we still found macroscopic flexion of the ankle joint of 5 ± 1° (Revised Supplementary Fig. 23c).Synchronously, we also have updated the revised Supplementary Video 2 with the rat's leg dangling in the air.
Revised Supplementary Fig. 23.a, Snapshot photo of a suspended and fixed ultrasound probe.b, Snapshot photo of the ultrasound coupling agent between the ultrasound probe and the rat skin.c, Representative images of the movement of the legs before and after stimulation.Average angular change of the ankle joint in response to stimulation.
4. For EEG signal, to the best of our knowledge, it can be measured at any time.How to prove the effect of ultrasound-induced electric stimulation?The comparative experiment should be carried out.
Response: Another good point.To fully evaluate the effect of ultrasound-induced electric stimulation, we used normal EEG (rats not receiving such stimulation) and US alone-induced EEG (rats not implanted with NeuroRing) as control groups.By comparing the results between these groups, we can assess the specific effects and benefits of ultrasound-induced electric stimulation.
Clearly, the intensity (frequency) of EEG signals were significantly enhanced in rats treated with ultrasound-induced electrical stimulation, but not significantly changed in rats treated with US alone without NeuroRing implantation (top two images in Fig. 4b, Revised Supplementary Fig. 26).This was more intuitively reflected from the quantified power spectral density derived from the EEG power spectrum, especially from 50 to 80 Hz, as marked by the black dashed box in the bottom image of revised Fig. 4b.(page 10, line 2 to 7 in revised manuscript) Also, in the original manuscript, from the data of neurohormone secretion and physiological markers as well as colitis treatment, compared with the control groups including US alone and sham groups, the US-induced electric stimulation shows significant advantages (Fig. 4c, Fig. 4e to q, Supplementary Figs.28 and 29).This further indirectly confirms the effect of ultrasoundinduced electric stimulation.Takahashi T, Nakagawa K, Tada S, Tsukamoto A. Low-energy shock waves evoke intracellular Ca(2+) increases independently of sonoporation.Sci Rep 9, 3218 (2019).6.For the colitis treatment, please tell more details about how to place the NeuroRing and connect the electrode and nerve.The Nerve is usually stimulated by DC electrical signal.In this experiment, no rectifier is utilized.How does it work?The record EEG signal is very weak and it is difficult to prove the effect of ultrasound-induced electric stimulation.Please explain it.

Response:
We thank the reviewer's comment.It is necessary to point out that the NeuroRing, which works in a wireless and electrodeless mode, is implanted by directly wrapping it around the splanchnic nerve extending from the S3 sacral nerve as shown in Fig. 4a.This is because the implant site can directly innervate the distal colon and rectum, reducing inflammation and pain in colitis. 1,2Due to the seamless connection between NeuroRing and nerve tissue, the electrical pulses generated by ultrasound are directly transmitted to the target nerve through the mediation of bodily fluids and tissues.To this end, we focused on describing the implantation details and working principles in the 'Methods' section as follows: 'SD rats (150 to 200 g) were fasted for 6 h prior to surgery, then anesthetized with inhalation of 0.8−1.5% isoflurane and maintained with 1.0% isoflurane.Before implantation, the NeuroRing was sterilized by ultraviolet light irradiation for 1 h.A skin incision was made at the coccyx, and the muscles divided to expose the sacral nerve and freeing it from surrounding tissue.The NeuroRing was implanted by wrapping it around the splanchnic nerve extending from the S3 sacral nerve and secured using biologic glue.Due to the seamless connection between NeuroRing and nerve tissue, the electrical pulses generated by ultrasound are directly transmitted to the target nerve through the mediation of bodily fluids and tissues.Muscle and skin were then closed with 4-0 and 3-0 nylon sutures, respectively.'(page 15, line 12-21 in revised manuscript) For the second comment, actually, the basic waveforms used in electrotherapy, including nerve stimulation, are generally divided into three groups: direct current (DC), pulsed DC, and alternating current (AC).Notably, in comparison with DC, AC electrotherapy is safer and better subjectively tolerated by the patient, and its DC component is always zero, which prevents chemical damage of the skin/tissue/organ. 7AC electrotherapy allows also for long-lasting applications in vivo.][9][10][11] In our study, under the excitation of ultrasound, NeuroRing can directly convert the sound waves into AC signals to stimulate nerve tissue without rectifiers (Fig. 2a-g).Immediately afterwards, these AC signals will act on nerve cells, inducing them to yield action potentials, and ultimately stimulate and modulate neural tissue (Fig. R1).
For the third comment, we recommend viewing revised Supplementary Video 3. to get a quick impression of the extraordinary features of our study.Once ultrasound activates the NeuroRing at the sacral nerve, the EEG signals change and intensify.This is further quantified by the quantified power spectral density derived from the EEG signal (lower curve, revised Fig. 4b).In the range of 50 to 80 Hz, the EEG of ultrasound-induced electric stimulation shows the greatest power density, which means the most active brain activity.This is consistent with the conclusion that NeuroRing shows significant advantages in treating colitis.

Fig. R1.
NeuroRing can be activated by ultrasound vibrations to yield electric impulses.On the basis of the resting potential, the electric impulses will stimulate the nerve cells in contact with NeuroRing, and trigger the local cells to produce a transmissible membrane potential fluctuation (i.e., action potential).After the action potential is generated, it is not limited to the local site stimulated, but propagates rapidly along the plasma membrane until the cells in the whole tissue produce an action potential in turn.Action potentials in nerve tissue play a central role in cell-cell communication.They propagate signals along the neuron's axon toward synaptic boutons which then connect with other neurons at synapses, or to motor cells or glands.Virtually all organs and their functions are regulated through such nerve impulses.

Reviewer #2
1. Since this is a passive implantable device, the accurate control of its stimulation parameters (e.g., strength) is very challenging considering alignment issues and tissue loss.It is also harder to scale it up to distributed implants for multi-site stimulation.

Response:
We highly appreciate the reviewer's professional comments and sharp scientific insights.2][3][4] For ultrasound receivers, there are still several main challenges: 1) interfacial losses due to acoustic impedance mismatches, 2) the reliance of receivers on rigid inorganic piezoelectric materials, and 3) the harsh efficiency penalty for transducerreceiver misalignment.Therefore, the required physical contact between devices and tissue, as well as losses due to impedance mismatches and misalignment, must always be considered during system design.These challenges can be addressed by novel functional materials with enhanced piezoelectric and acoustic properties.By decoupling the above three issues, we designed and fabricated a ring-shaped soft fibrous ferroelectret (i.e., NeuroRing).It facilitates precise control of stimulation parameters (e.g., intensity) and modulation of nerves.Details are as follows: 1) Fibrous non-woven fabric designed for reducing acoustic impedance.A mismatch in acoustic impedance is common between transducer materials and the tissue because these materials have different average acoustic velocity and densities.When two materials have large differences between acoustic impedance ultrasound signals are reflected at the interface, leading to wave reflection and reduced powering efficiency. 5For example, water has an acoustic impedance of 1.5 MRayl, and human soft tissue demonstrates an acoustic impedance of 1.63 MRayl. 6The typical values ceramics are >30 MRayl for bulk or plate-type piezoelectric and <3 MRayl for polymeric piezoelectric. 7,8Clearly, soft polymeric piezoelectric materials can effectively reduce acoustic impedance.On the other hand, many studies have found that fiber polymers have lower acoustic impedance and can even resolve acoustic impedance mismatches. 9,10This has been widely verified in nature, for example, in the human auditory system, the tympanic membrane is responsible for resolving the acoustic impedance mismatch between the air of the ear canal and the fluid of the inner ear. 11Therefore, we used electrospun fibers-based non-woven fabrics to construct the NeuroRing.
2) High-performance ferroelectret with tissue-like mechanical modulus.3][14][15] However, they are mechanically mismatched to soft neural tissue, which causes insertion-related lesions, inflammation reactions, and even neuronal apoptosis, ultimately even leading to therapeutic failure. 16,17Although much effort has attempted to overcome these limitations by developing piezoelectric composite materials that combine piezoelectric ceramics and polymers, they have been unsuccessful as either piezoelectricity or flexibility and processability are compromised.9][20] To address these issues, we introduce a porous ferroelectret structure by utilizing the cavitation effect between inorganic particles and soft electrospun PVDF matrix, which can obtain additional electric dipoles at the surface of the material and inside it, thus enhancing the electrical performance output while maintaining flexibility.2][23] Meanwhile, Young's modulus value is estimated to be 7.6 MPa which is in the same order as soft tissue. 24This modulus value is four to six orders of magnitude lower than that of commonly used piezoelectric ceramics such as PZT and BaTiO3, and its piezoelectric coefficient was 1 to 2 orders of magnitude higher than that of piezoelectric polymers with similar modulus value (Fig. 1f, Revised Supplementary Table 1).These two points ensure that our NeuroRing attaches perfectly to soft nerve tissue and converts ultrasound pulses into electrical pulses efficiently.
3) Ring-shaped design for improving alignment.A disadvantage of this approach is that ultrasonic waves propagate directionally.As a result, slight misalignments and misorientations between the external transducer and the implanted receiver lead to reductions in coupling efficiency. 25The positioning and form (e.g., pulse frequency) of the ultrasound transmitter are critical for effective power delivery, which can be adjusted based on the location and positioning of the ultrasound receiver. 26However, for implanted ultrasound receivers, especially small ones (millimeter size), regulating external ultrasound transmitters is obviously difficult and complex.Fortunately, research has found that designing novel receiver geometries, such as cylinders of piezoelectric materials, holds promise in increasing tolerance to rotational misalignment efforts. 27,28Considering that the application scenario of our study is peripheral nerve modulation, the high-performance fibrous ferroelectret film is designed in a ring shape that is able to tightly wrap around the nerve.We measured the ultrasound intensity applied to the ring-shaped devices and found that they were not affected by rotational misalignment.When excited by 0.5 W cm −2 ultrasound pulses with a frequency of 1 MHz, normal incident ultrasound produces a similar ultrasound intensity (0.2 ± 0.01 W cm −2 ) irrespective of ultrasound transmitter rotation (black curve in revised Supplementary Fig. 13b).At the same time, we simultaneously recorded the voltage outputs and found that they were also unaffected by rotational misalignment as long as the distance between the ultrasound probe and the fiber membrane was the same (blue curve in revised Supplementary Fig. 13b).This is crucial for precise peripheral neuromodulation.(page 5, line 9 to 12 in revised manuscript) Thanks to these optimizations, our fibrous ferroelectret non-woven fabric were able to efficiently convert ultrasound vibrations into electric impulses.It can even yield a voltage output of more than 250 mV at a depth of 10 cm under layered tissues including skin, fat and muscle, which was sufficient to stimulate peripheral nervous system in humans from a few millimeters to more than ten centimeters (covering most of the peripheral nerves).Besides that, when our NeuroRing is implanted into the body of rats, despite some deflection of the ultrasonic transmitter, the nerves can still be effectively regulated, which is further reflected on the EEG signals (revised Supplementary Fig. 27).(page 10, line 9 to 11 in revised manuscript) Indeed, multi-site stimulation is a drawback of our technology, and we appreciate the reviewer's constructive reminder.This promotes a more in-depth and detailed engineering design of our NeuroRing in the application of neurostimulation.The possible feasible solutions we envision are: 1) Reducing the width of the NeuroRing and implanting multiple devices in parallel in the same area; 2) Designing multiple parallel arranged small unit NeuroRings on a single device through hollowed-out pattern method.In future research, we will further validate these two hypotheses.We hope that through these two designs, our NeuroRing has the potential to expand into multi-site stimulation applications in the human body in the future.Revised Supplementary Fig. 13.b, The relationship between ultrasound intensity on ring-shaped ferroelectret film and the deflection angle of ultrasound probe.

Revised
Revised Supplementary Fig. 27.After the ultrasonic incidence angle was deflected by 60 °, the nerve was still activated, which was reflected in the unchanged EEG signal compared with the vertical excitation.
2. The main novelty of this work is on developing new soft ultrasonic materials.Its application for nerve stimulation is less significant considering the available devices.Therefore, the authors must do a better job in providing a comprehensive comparison of the performance of this material with the relevant state-of-the-art materials.

Response:
We thank the reviewer's comment and constructive suggestion.First of all, we agree with the reviewer's point of view that the novelty of this work is the design and preparation of new soft electromechanical coupling materials for ultrasound receivers.To comprehensively and objectively prove the superiority of our materials, we conducted a thorough literature review and comparisons with the relevant state-of-the-art materials, covering ceramics, polymers and their composites (Fig. 1f).For details, please find them in revised Supplementary Table 1.2][3][4][5] It is expected to be used in vivo as an adjunct or alternative to drugs to treat neurological disorders and diseases via stimulating the peripheral nervous system on demand triggered by ultrasound.7][8][9] They are mechanically mismatched to soft neural tissue, which causes insertion-related lesions, inflammation reactions, and even neuronal apoptosis, ultimately even leading to therapeutic failure. 10,11Many efforts have attempted to develop piezoelectric composites combining inorganic particles and polymers to exploit their advantages and overcome their limitations.However, their properties, such as piezoelectricity and flexibility, are still far below expectations.The 'rule of mixture' is often used to predict the properties of ideal composites, but it appears to have been broken in the design and development of functional piezoelectric hybrids, with either piezoelectricity or flexibility and processability being compromised.The problem is that even the tight bond between the inorganic particles and the polymer matrix cannot improve the material's properties substantially enough for practical applications while retaining its flexibility.For example, as reported in Nature in 2022, Yan et al. developed a ferroelectret composed of a mixture of PVDF-TrFE and BaTiO3 with a high piezoelectric coefficient up to 46 pC N -1 , but at the expense of the flexibility of the polymer matrix. 12It is still mechanically mismatched to soft neural tissue.4][15] Innovatively, we introduce a porous ferroelectret structure by utilizing the cavitation effect between inorganic particles and soft electrospun PVDF matrix, which can obtain additional electric dipoles at the surface of the material and inside it, thus enhancing the electrical performance output while maintaining flexibility.7][18] Meanwhile, Young's modulus value is estimated to be 7.6 MPa which is in the same order as soft tissue. 19This modulus value is four to six orders of magnitude lower than that of commonly used piezoelectric ceramics such as PZT and BaTiO3, and its piezoelectric coefficient was 1 to 2 orders of magnitude higher than that of polymers with similar modulus value.Therefore, taken together, we break down the common barrier wall in piezoelectric mixtures of inorganic particles and polymer matrix-the incompatibility of piezoelectricity and flexibility.Our materials achieve the trade-off between flexibility and piezoelectricity, maintaining excellent flexibility while also achieving extremely high piezoelectricity.
On the other hand, in addition to innovation of materials, it is necessary to point out that our NeuroRing also has significant advantages over currently available neurostimulation devices.Our paper presents a novel approach to peripheral neuromodulation using a soft ring-shaped ultrasound receiver.This innovative device offers several advantages over existing technologies, including high mechanical conformability to soft nerve tissue, wireless operation, and reduced risk of injury and inflammation.The development of tissue-matched ultrasound receivers is not only important for advancing our understanding of neurological disorders but also for improving the lives of millions of people around the world who suffer from these conditions.By supporting this technology, we can help to bring about a new era of targeted treatments for neurological disorders.
For details, please refer to the response to comment 1.For the stability study, we placed ferroelectret materials soaked in phosphate-buffered saline in a thermostat gas bath vibrator (CHA-SA, China) at 37 °C for 6 months.During this period, we tested the piezoelectric output of the ferroelectret materials every month.Before testing, these materials were washed three times with absolute ethanol to remove residual buffer, then immersed in absolute ethanol and excited using an ultrasonic generator (1 MHz, 0.5 W cm -2 ).The test parameters are the same as the original manuscript.Simply, the distance between the ultrasonic probe and the ferroelectret film is 10 mm.The test device operated in single-electrode mode using an aluminum foil as the electrode, and a copper wire conducting the charges, as shown in Fig. 2a.

Revised
The results were presented in revised Supplementary Fig. 13a.It can be seen that the ferroelectret materials have excellent stability, and the output peak-to-peak voltage is maintained at 6 ± 1 V with almost no change.(Page 5, line 7 to 9 in revised manuscript) As for the biocompatibility study, before implantation in rats, we first performed cytocompatibility testing in vitro.Neural stem cells were cultured on the ferroelectret materials and petri dishes (gold standard).After 3 days of culture, the expression level of Ki67 was assessed (Fig. R2a).Ki67 is a related antigen of proliferated cells and can be used as marker of proliferation ability.It can be seen that the cell proliferation on the ferroelectret materials did not exhibit significant differences compared to the culture dishes (Fig. R2b).These data provide preliminary confirmation that our ferroelectret materials were biocompatible and nontoxic.Then, to further verify the long-term biocompatibility in vivo, the ferroelectret materials were implanted into the gastrocnemius muscle area and around the sciatic nerve of the rats for nearly 6 months (Fig. R3a).This region was associated with greater muscle rhythmicity, which could diagnose the potential infections and necrosis in surrounding tissues and evaluate the mechanical stability of the materials in vivo.The implants were removed at the 1st, 3rd, 6th and 24th weeks after implantation.Histological analyses were performed by staining prepared tissue slides with hematoxylin and eosin (Fig. R3b).The histological images of the implantation area showed a very mild immune reaction without significant presence of inflammation and cellular toxicity.Fibrosis and activated macrophages were found in the 1st week, improved from week 3, and reduced to the normal level at week 6, and continued until the 24th week.These data confirmed long-term biocompatibility of our ferroelectret materials.
Revised Supplementary Fig. 13.a, Comparison of the output voltage before and after the ferroelectret fibers being immersed in a PBS solution at 37 °C for 6 months.4. Measurement result in Fig. 2: The external transducer was driven at 1 MHz?Why was this frequency chosen?Is the implanted flexible piezoelectric device resonating?Its dimension is 5 mm which is much lower than 1 MHz.

Response:
We thank the reviewer's professional comment.Yes, the measurement results in Fig. 2 were taken at an ultrasonic frequency of 1 MHz.We selected 1 MHz after comprehensive consideration of various factors, such as biosafety, spatial resolution and electromechanical coupling efficiency, etc.For now, actually, many studies choose ultrasound with low frequencies < 1 MHz.This is because low frequencies can improve the efficiency of energy transfer by reducing tissue-mediated attenuation, [1][2][3] compensating to a certain extent the low piezoelectric performance of implanted ultrasound receivers.However, low frequencies often cause deleterious tissue effects, such as cavitation and inevitable heating, [4][5][6] which prone to cause apoptosis, tissue collapse, or even necrosis, and thus disrupt the physiological function of normal tissues.Also, low frequencies accompanied by low resolution will result in a large focal region, making it difficult to accurately locate the precise stimulation area. 2 This is a critical issue, especially for micronsized peripheral nerves.According to the principle of acoustics, high-frequency ultrasound has the ability to generate small focal region, thereby improving the accuracy of stimulation. 7Narrowing the stimulation area with high-frequency ultrasound will provide good opportunities to expand its application. 8,9For example, focused ultrasound transducer with 1 MHz frequency will generate a focal width of about 4.3 mm.What cannot be ignored is that at high frequencies, especially above 10 MHz, the absorption of ultrasound by biological tissues becomes substantial, resulting in extremely severe ultrasound attenuation. 10,113][14][15][16] Under this premise, as long as our ultrasonic receiver has sufficiently high piezoelectric performance, we choose a higher frequency of 1 MHz.In fact, many studies do the same, 1 MHz was chosen as the driving frequency. 1,17 for whether our ultrasonic receiver resonates with ultrasonic waves with a frequency of 1 MHz, we tested and counted the voltage output of ferroelectrets driven by ultrasound at frequencies ranging from 200 kHz to 1400 kHz (Fig. R4).It can be inferred that the resonant frequency of our fibrous ferroelectret is around 700 kHz due to the high voltage output at this frequency.Thanks to the high performance of our ferroelectric materials, it is ensured that the ultrasound receiver can generate sufficient electrical stimulation pulses at non-resonant frequencies.Of course, we appreciate the constructive questions from the reviewers, which inspired us to customize electromechanical coupling materials that resonate with high-frequency ultrasound (>1 MPa) in future research to further improve piezoelectric properties and biocompatibility. 5. Measurement result in Fig. 2: The authors claim that these voltages are enough for neural stimulation.But these are 1 MHz pulses.For successful stimulation, kHz pulses are often applied to a nerve.Also, the current injected to the nerve is important (not the voltage necessary).What is the injected current (or alternatively the electric field) applied to the nerve in these conditions?
Response: Good point.Although pulses with frequencies of MHz pulses can be effectively used for neural regulation, the specific mechanism is still not clearly elucidated. 1,6We are very grateful for the reviewer's comment, which is also a question we have been thinking about.
8][9] Our study is consistent with this hypothesis that ultrasoundinduced electrical pulses promoted the opening of Ca 2+ channels in SH-SY5Y-derived neuron-like cells (Fig. 2h to k).
We agree with the reviewer that the current injected into the nerve is also important.To this end, we supplemented the current-related data.The test method is the same as the corresponding voltage test method under pork tissue as shown in Fig. 2c and d.We used a current probe (CP6510, Siglent) for current collection.Simply, the packaged non-woven ferroelectret fabrics (dimension of 5 mm by 10 mm) were inserted into porcine tissue for ex vivo testing.As the implantation depth increases, the current shows the same trend as the voltage (Revised Supplementary Fig. 15c).Regarding the injection current applied to the nerve, considering that both the sciatic and sacral nerves were located about 1 cm beneath the skin of rats, we therefore studied the current output of ferroelectrets with different sizes at 1cm in pork tissue.We found a positive correlation between the size of ferroelectrets and their current output in the range of 1 mm 2 to 50 mm 2 (Revised Supplementary Fig. 15d).For the sciatic nerve, the injection current is 13 ± 4 nA owing to the ferroelectret with an area of about 2 mm 2 , while for the sacral visceral nerve, it is 8 ± 4 nA owing to the ferroelectret with an area of about 1 mm 2 .
6. Measurement result in Fig. 2b: At what frequency did you pulse the 1 MHz ultrasound transducer?What is the source of large background voltage when the pulse is zero?
Response: We thank the reviewer's comment.We used a mains frequency of 50 Hz to power the 1 MHz ultrasound transducer.It is necessary to point out that the large background of the voltage curve is present in the entire waveform, not just when the ultrasound stops.The source of the background of the voltage curve in Fig. 2b (left) may be a variety of signals, such as noise, electromagnetic interference at 50 Hz, and overlapping signals of high-frequency electrical signals at the low sampling rate of the oscilloscope.To confirm the source of the background signal, we simply replaced the ferroelectret film with a non-piezoelectric fibrous polylactic acid film.Subsequently, the same test method as Fig. 2b is used for signal collection (Fig. R5).We found that the noise and electromagnetic interference signals were much smaller than the background signals in Fig. 2b.Therefore, we can conclude that the background source of the voltage curve is mainly the overlap of the electrical signal of the ferroelectrets.This may be due to the fact that the liquid environment in which the ferroelectret film is located is still in a weak vibration state even if the ultrasound pulse wave stops.7. Since the ultrasound sonication (thereby electrical pulses) was at 1 MHz, what was the underlying mechanism for neuromodulation in in vivo tests/results in Fig. 3 and 4. Also, what is the estimated voltage on the implant in these tests?
Response: Good point.We highly appreciate the reviewer's professional comment.7][8] Our study is consistent with this hypothesis that ultrasound-induced electrical pulses promoted the opening of Ca 2+ channels in SH-SY5Y-derived neuron-like cells (Fig. 2h to k).
Regarding the estimated voltage applied to neural tissue, we need to take into account the size and depth of the implanted ferroelectret.Therefore, considering that both the sciatic and sacral nerves were located about 1 cm beneath the skin of rats, we studied the voltage output of ferroelectrets with different sizes at 1cm in pork tissue.We found a positive correlation between the size of ferroelectrets and their voltage output in the range of 1 mm 2 to 50 mm 2 (Revised Supplementary Fig. 15e).For the sciatic nerve in Fig. 3, the estimated voltage is 0.5 ± 0.1 V owing to the ferroelectret with an area of about 2 mm 2 .For the sacral splanchnic nerve in Fig. 4, the estimated voltage is 0.2 ± 0.1 V owing to the ferroelectret with an area of about 1 mm 2 .
Reviewer #3 1.The first observation that drives the decision on this article relates to novelty of the work done.The foundation of the paper revolves around the use of PVDF/ZnO film obtained through electrospinning as the ferroelectret.However, it is well-known that electrospinning can prepare PVDF nanofibers (e.g., Sensors (Basel).2020 Sep; 20(18): 5214).Incorporating nanoparticles like ZnO is not uncommon in such studies.Additionally, this reviewer has doubts about the claim that their electrospun PVDF/ZnO can be considered a ferroelectret device.Firstly, their voids appear to be very small (Fig. 1d), which, according to calculations and previous experiments by ferroelectret nanogenerators (FENG) researchers, may not generate sufficient piezoelectric effects.

Response:
We thank the reviewer's comments.We respectfully disagree with the reviewer at this point, and we appreciate this opportunity to better state the novelty and significance of this paper in the field of piezoelectric materials, soft functional materials, and biomedical devices.Indeed, PVDF nanofibers and the incorporation of nanoparticles including ZnO and BaTO3 into the fibers have long been reported, 1,2 but the fact is that their piezoelectricity is still low, limiting their electric physiotherapy as effective receivers unless flexibility is sacrificed.Innovatively, we utilize the cavitation effect to introduce ferroelectret pore structure into the soft electrospun PVDF fiber matrix (please find details from Supplementary Fig. 1), which can obtain additional electric dipoles at the surface of the material and inside it, thus enhancing the electrical performance output.This theory that the electret effect occurs in the gap between ZnO and PVDF is reasonably inferred based on a large amount of experimental data and literature research.Specifically, The d33 of the ferroelectret fibers is the highest (56 ± 2 pC N −1 ), which is more than twice the airblowing PVDF fibers (22 ± 2 pC N −1 ) or PVDF/ZnO composite fibers (25 ± 2 pC N −1 ) (Supplementary Fig. 6).To understand this enhancement, we studied the polymer chain's orientation and crystallinity.First, we performed thermogravimetric analysis on PVDF fibers, airblowing fibers, PVDF/ZnO composite fibers, and ferroelectret fibers (Supplementary Fig. 5a).
All fibers experienced obvious weight loss at 400°C.At this stage, the PVDF molecular chains were decomposed to remove H-F in the molecules.Among them, the decomposition rate of ferroelectret fibers is relatively the lowest, indicating its high crystallinity.Crystallinity was further quantified by 1D XRD (Supplementary Fig. 5b).We calculated the crystallinity of the PVDF fibers, air-blowing fibers, PVDF/ZnO composite fibers, and ferroelectret fibers to be 27.5%, 52.1%, 66.3%, and 68.9%, respectively.According to references, [3][4][5] the enhanced d33 can be attributed to the existence of the oriented amorphous fraction that exhibits improved dipole mobility and better chain alignment after the air-blowing process, enhancing the piezoelectric properties of the fiber.Therefore, we used 2D XRD to further study the orientation of the polymer chain in the fiber.As shown in Supplementary Fig. 5c, the results reveal that the air-blowing electrospin process can align the polymer chains along the fiber axis direction.The orientation degrees of fibers are quantified using Herman's orientation factor.We calculated the orientation degrees of the PVDF fibers, air-blowing fibers, PVDF/ZnO composite fibers, and ferroelectret fibers, using Herman's orientation factor to be 0.78, 0.83, 0.80, and 0.81, respectively.Overall, the comparable crystallinities between the PVDF/ZnO composite fibers and ferroelectret fibers as well as the comparable orientation degrees between the air-blowing fibers and ferroelectret fibers suggest that the enhanced value of the piezoelectric coefficient is driven neither primarily by a flow induced orientation effect nor by the crystallinity, but rather by another mechanism.Therefore, there must be a synergistic effect from both the PVDF matrix and ZnO particles.We thus performed TEM characterization on the air-blowing PVDF/ZnO fiber (Fig. 1d, Supplementary Fig. 4) and observed cavitation on both sides of the ZnO particles, with the cavities elongated axially along the fiber (i.e., gas blowing direction).The existence of cavities in the vicinity of the ZnO particles, which are found only in the air-blowing fibers and not in the general electrospin composite fibers, leads us to suggest that cavitation in the drawn composite fiber is the major contributor.
From this observation, a mechanism of enhanced piezoelectricity due to the dimensional effect for the air-blowing PVDF/ZnO fiber is proposed (Fig. 1c, Supplementary Fig. 1).With initial electrospining, PVDF undergoes solidification and crystallization.Upon air drawing of the crystallized sample, cavitation takes place around the ZnO particles, forming horizontal pores on the two sides.After electric poling during electrospinning process, ferroelectric domains in PVDF are polarized, generating an electret effect.When deforming the poled PVDF/ZnO fiber during the direct piezoelectric test, the pore volume changes, creating a dimensional effect for enhanced piezoelectricity.That is, the change of dipole density by changing the pore volume induces significantly improved piezoelectricity.Similar PVDF foam electret has been reported to show significantly enhanced piezoelectric performance due to the porous structure. 6In this sense, the drawing-aligned oriented amorphous fraction and the increased dielectric constant of the composite have a much weaker contribution to the enhanced d33. 4,7It should be noted that conventional foamed ferroelectrets are prone to depolarize and lose their piezoelectric properties rapidly.In this study, for the first time, we have developed a new class of stable PVDF-based ferroelectret devices by creating cavitation using one-step laminar-flow-assisted electrospinning method.This approach paves a novel route towards a new paradigm of fiber-based, highperformance ferroelectret transducers.
In conclusion, the high performance of the thermally drawn PVDF/ZnO fiber can be attributed to cavitation between ZnO particles and the PVDF fiber matrix, and the well-aligned orientated amorphous fraction, which further increases the piezoelectric performance.
As for the reviewer's concern about the small size of the void (200 nm of radial length, 100 nm of axial length), it is unfounded considering the size of the electron itself (diameter < 10 −9 nm) and its storage.Obviously, our design does not have the problem of too small gaps.0][11][12] However, large pore sizes can easily depolarize and rapidly lose their piezoelectric properties.Our ferroelectric electrets exhibit stable performance that can last for > 6 months on end (Revised Supplementary Fig. 13a).For details, please refer to the response to the second reviewer's comment 3.
2-3.Along this same line, where are the current plots?Did the authors try to characterize output short circuit current from these devices?Did the authors consider the matching impedance between the device and the instrument used to measure the generated voltage?Obtaining real electromechanical measurements from ferroelectrets is not a trivial task, and this reviewer believes that these experiments/studies will generate questions about the electrical voltage output that they are measuring -which could be due to sources other than the ferroelectret.

Response:
We thank the reviewer's comment.In the original manuscript, we did not measure short-circuit currents since our goal is to use the local piezopotential yielded from ultrasound receiver to stimulate and regulate nerve tissues, rather than for energy collection or storage.Similarly, since our application aims to stimulate nerves with local piezopotentials, we tend to directly use a voltage probe with 40 MΩ input impedance to collect open-circuit voltage without considering matching impedance, as described in the 'Methods' section of the original manuscript.
(page 13, lines 29 and 30 in revised manuscript) Of course, for the sake of rigor, per the reviewer's request, we conducted experiments on current collection and resistance matching.We use a current probe (CP6510, Siglent) for current collection, and the testing method is the same as the voltage collection in Fig. 2b.The peak-to-peak shortcircuit current is approximately 240 nA (Fig. R7a).As shown in Fig. R7b, the change of output voltage (blue) and current (red) for the ferroelectret device in an external load resistance range from 100 Ω to 100 MΩ.As the load resistance increases, the output voltage keeps raising until saturation at high resistance (>10 MΩ).On the contrary, the current continuously decreases.
As for the source of voltage, since the entire device is operated in a liquid or biological tissue environment, we can basically exclude the voltage generated by triboelectric effects.This is further verified through experiments on exposed fibrous ferroelectrets in biological tissue fluids (Fig. 2e  and f).Furthermore, to eliminate electromagnetic and noise interferences, we simply replaced the ferroelectret film with a non-piezoelectric fibrous polylactic acid film.Subsequently, the same test method as Fig. 2b is used for signal collection.From Fig. R5, we found that the noise and electromagnetic interference signals were much smaller than the voltage signals in Fig. 2b.For details, please refer to the response to the second reviewer's comment 6.Therefore, we conclude that the collected voltage mainly comes from the piezoelectric signal of the ferroelectrets.3. Finally, although their application may appear fancy, it takes a quick look to the references and relevant prior art to realize that this is also not a novel concept -this reviewer has personally read this same application roughly 5 years ago.Lastly, it's disheartening to note that they not only failed to cite any recent relevant work (including a very recent comprehensive review article on ferro electrets and their applications), but also neglected to reference important studies by other wellknown researchers in the field of ferroelectrets.

Response:
We thank the reviewer's comment.Regarding the selection of exemplary applications, please allow us to politely elaborate on the reasons why we chose to treat colitis by stimulating the sacral nerve, not for fancy purposes, but based on the shortcomings or deficiencies of the existing treatment protocols.3][4][5] However, it cannot be ignored that most of these reports inevitably use rigid electrodes, long wires, and external power sources, which are not suitable for long-term and clinical applications.][9][10] Besides, mechanical mismatch at the rigid electrode-soft neural tissue interface can cause trauma and insertion-related lesions, inflammation reactions, and even neuronal apoptosis, ultimately leading to therapeutic failure. 7,11,12We provide a wireless, leadless, and battery-free treatment strategy that precisely regulates and alleviates colitis by triggering stimulation of peripheral nerves with ultrasound pulses.Besides, our device exhibits high mechanical conformability to dynamic motion nerve tissue and can wrap around the target peripheral nerve without affecting normal development and movement.Of course, our study of sacral nerve stimulation for colitis is just a demonstration of its application, which can provide a solid foundation for electromodulation of peripheral nerves to treat disease.
As for the comment we did not cite the latest ferroelectret-related research, we believe it is unfounded.In fact, we have conducted extensive analysis and citation of existing electrets, especially those based on PVDF piezoelectric materials, in the original manuscript (Revised Supplementary Table 1).As is well known, depending on the charge carrier type, electrets are divided into charge electrets and dipole electrets.The electret charges are either real excess charges (charge electrets) or result from oriented dipoles (dipole electrets). 13Ferroelectrets are a member of the electret family (charge electrets) based on nonpolar polymers with a porous foam structure with open or closed cells where the internal surfaces carry positive and negative charges.Foamed ferroelectrets have been widely fabricated based on polyethylene and polypropylene polymers because of their good insulating properties.However, their poor stability limits their application as they are prone to depolarize and lose their piezoelectric properties rapidly. 14Notably, as a representative of semicrystalline dipole electrets, PVDF was found to exhibit considerable piezoelectric and charge stability as early as the 1960s and early 1970s. 15,16Therefore, in this study, for the first time, we introduced ferroelectret pore structure by using a one-step laminar-flowassisted electrospinning method, further increasing the number of electric dipoles in PVDF dipole electrets, and thus developing a new class of stable fibrous PVDF-based ferroelectret devices.This approach paves a novel route towards a new paradigm of high-performance and stable soft ferroelectret transducers.For details, please refer to the description of the Supplementary Fig. 6 in Supplementary Materials.(pages S8 to S10 in Supplementary Materials)

Fig. R2. a ,
Fig. R2.a, Representative images of Ki67 (proliferation marker)-marked cells cultured on ferroelectret materials and petri dish in proliferation medium for 3 days.b, Relative Ki67 expression levels of cells on ferroelectret materials and petri dish.n = 5 for Ki67 expression level statistics.

Fig. R5 .
Fig. R5.Voltage output measured in ethanol at a distance of 10 mm from an ultrasound probe to the non-piezoelectric polycaprolactone film, with an ultrasound setup of 1 MHz and 0.5 W cm −2 .

Fig. R7. a ,
Fig. R7.a, Current output measured in ethanol at a distance of 10 mm from an US probe to the ferroelectret, with a US setup of 1 MHz and 0.5 W cm −2 .b, The output voltage, and current of the ferroelectret under different load conditions measured in ethanol at 10 mm from an ultrasound probe to the ferroelectret film, with an ultrasound setup of 1 MHz and 0.5 W cm −2 .

Table 1 .
Supplementary Comparison of d33 piezoelectric coefficient between ceramics, polymers, and state-of-the-art composites.

Table 1 .
Supplementary Comparison of d33 piezoelectric coefficient between ceramics, polymers, and state-of-the-art composites.We thank the reviewer's comment.Per the reviewer's request, we provided stability and biocompatibility data for ferroelectret materials.Actually, prior to being used as a NeuroRing for neuromodulation, our ferroelectret materials underwent stability testing and a 6-month subcutaneous biocompatibility testing.Our ferroelectret materials demonstrate excellent long-term stability and biocompatibility.Specifically, [1][2][3][4][5]For example, TaejeongKimet al. used piezoelectric particles driven by ultrasound at a frequency of 1 MHz for deep brain stimulation in the treatment of Parkinson's disease. 1 Eli J. Curry et al. used 1MHz ultrasound triggered piezoelectric materials for deep brain stimulation to open the blood-brain barrier. 2 Joshua C. Chen et al. successfully activated the sciatic nerve of rats using a 1.25 MHz magnetoelectric pulse.